Methods of Detecting Antibodies Specific for Denatured HLA Antigents

ABSTRACT

The invention is directed to methods of screening for HLA antibodies comprising detecting antibodies specific for native HLA antigens and denatured HLA antigens. The invention also provides for methods of removing antibodies specific for denatured HLA antigens or antibodies specific for native HLA antigens from a serum sample. In addition, the invention also provides for method of predicting whether a transplant recipient has an increased risk for rejecting the transplanted organ.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 12/163,524, filed Jun. 27, 2008, which claimspriority benefit of U.S. Provisional Patent Application No. 60/947,872,filed Jul. 3, 2007 and U.S. Provisional Patent Application No.60/949,806, filed Jul. 13, 2007, all of which are incorporated byreference herein in their entirety.

FIELD OF INVENTION

The invention is directed to methods of screening for HLA antibodiescomprising detecting antibodies specific for native HLA antigens anddenatured HLA antigens. Native class I HLA antigens are non-covalentlyassociated with β2-microglobulin and denatured HLA class I antigens aredisassociated from β2-microglobulin. Native HLA class II antigens arenon-covalently bound hetero-dimers and denatured HLA class II antigensare monomers. The invention also provides for methods of removingantibodies specific for denatured HLA antigens or antibodies specificfor native HLA antigens from a serum sample. In addition, the inventionprovides for methods of predicting whether a transplant recipient has anincreased risk for rejection of the transplanted organ or has anincreased risk for developing graft vs. host disease.

BACKGROUND

Transplant rejection occurs when the immune system of the recipient of atransplant, particularly antibodies produced by the recipient, attacksthe transplanted organ or tissue. The recipient's immune systemrecognizes the transplanted organ as foreign tissue and attempts todestroy it. Rejection also occurs when the transplanted organ comprisesthe donor's lymphocytes or progenitor stem cells, which may generate animmune response to the recipient tissues such as graft vs. host disease.Chronic rejection is a term used to describe all long term loss offunction in organ transplants associated with chronic alloreactiveimmune response. Long term chronic rejection usually leads to a need fora new transplanted organ about a decade after the initial transplant.Human leukocyte antigens (HLA) are one type of molecules within atransplanted organ in which the recipient's immune system attacks thatcauses a transplant rejection.

An HLA class I molecule consists of a 45-kDa glycoprotein (heavy chain)non-covalently associated with a 12-kDa polypeptide, β2-microglobulin(β2m). Association of β2m with newly synthesized class I heavy chains isrequired in order for the HLA molecule to transport and present thepeptide (Krangel et al., Cell 18: 979, 1979). However, β2m free class Iheavy chains were identified on activated T lymphocytes (Schnabl et al.,J. Exp. Med. 171:1431, 1990) and other cell surfaces (Bix & Raulet, J.Exp. Med. 176(3) 829-34, 1992). Properly conformed β2m free class Iheavy chains were identified on the cells and were believed to havefunctional importance. β2m can be dissociated from a HLA class I complexon a cell surface by acid treatment (Sugawara et al., J. Immunol.Methods, 100(1-2):83-90, 1987). β2m can also be dissociated from HLAClass I complex coated on microbeads using the similar method of low pHtreatment. (Pei et al. Visuals Clinical Histocompatability Workshop2000, 9-10). Those β2m-free HLA heavy chains are referred to as“denatured antigens.” Antibodies against denatured class I HLA antigenshave been detected in human sera from non-sensitized donors, however,they have not been well studied and currently the clinical significanceof these antibodies is unclear.

HLA class II molecules are heterodimers formed by noncovalent linkage oftwo glycosylated polypeptide chains referred to as alpha and betachains. The α subunit is 33 kDa and the β subunit is 28 kDa, and bothchains are transmembrane polypeptides that have the same overallstructure. The invariable a chain is encoded by the DRA HLA gene andthis chain binds various β chains encoded by the DRB HLA genes. Inaddition, the DP and Dq HLA gene families each have one gene thatencodes an α chain and a β chain. (Reviewed in Choo, Yonsei Med. J. 48:11-23, 2007).

Cell-based assays are the most widely accepted assay for cross-matchingHLA antigens, and these assays are used to determine if a recipient hasantibodies in their serum that are cytotoxic to the lymphocytes of aprospective donor. The donor cells for the cross-match assay arecollected from either blood or spleen and must be alive, while therecipient serum can be frozen prior to the assay. Exemplary standardprocedures for cross-matching include complement-dependent cytotoxicitytest (CDC), CDC with antiglobulin augmentation (AHG) and flow CytometryCross-match (FC) (Noreen, The American Society for Histocompatibilityand Immunogenetics Laboratory Manual, 3rd Ed, I.C.1.1-I.C.I.13).

As the number of identified HLA antigens is continuously increasing, acell panel must be extremely large to screen for all known HLA antigensin a single assay. In addition, the cell-based assays may be neglectingclinically important antigens as about 50% of transplanted organs willbe chronically rejected over a long period of time (known as “long temchronic organ rejection”). Cell-based assays are also limited by thefact that the target cells isolated under the normal condition bear onlyHLA antigens in their native conformation such as β2m-associated class Iantigens (Sugawara et al., J. Immunol. Methods, 100(1-2):83-90, 1987)and hetero-dimeric class II antigens. Thus, the cell-based assays willnot detect antibodies specific for denatured HLA antigens.

The development of solid-phase HLA antibody detection assays, such asbead-based assays, allow for screening of many HLA antigens in oneassay. Therefore, many laboratories are using solid-phase HLA antibodydetection assays and virtual cross-matching assays in addition tocell-based assays. “Virtual cross-matching” is a procedure that predictsthe result of a cell-based cross-match assay. Virtual cross-matching iscarried out by comparing the donor antigen profile (donor tissue typing)and the recipient HLA antibody profile as determined by solid-phaseassays described herein. For example, some laboratories first screen therecipient sera using a solid-phase assay to determine potential donorsmatches and subsequently use a cell-based assay for the cross matchanalysis of the donor cells and recipient sera. With the currentscreening procedures, sera that is positive for antibodies specific fordonor HLA antigens in both the solid-phase assay and the cell-basedassay is certain not to be an appropriate match for transplant. However,if the recipient sera is positive for antibodies specific for the donorHLA antibodies in the solid-phase assay but negative in the cell-basedassay, there is uncertainty whether this sera is false-positive forHLA-antibodies and whether the tissue can be used for transplant.

One of the advantages of determining the HLA antibody profile usingsolid-phase assays is that this information can be stored for thetransplant recipients. Therefore, biological samples do not need to beacquired at the same time or need to be in the same location foranalysis. The HLA antibody profiles will allow for analysis before anorgan is shipped for transplant. In addition, solid-phase HLA antigenanalysis can screen many more donors and recipient pairs and may screenmany more antigens at one time or in fewer assays. In addition, theseassays may decrease the incidence in which a positive cross-match organis shipped for transplant before the determination.

As increasing numbers of sera samples were tested for HLA antibodies,there was a significant increase in the number of samples that werepositive using the solid-phase assay but negative using cell-basedcross-match assays. In addition, antibodies to denatured class I HLAantigens are known to be present in the human sera and particularly someuntransfused males have been shown to have HLA antibodies in their sera.

It is known that acid treated cells, which dissociated βm2 from thesurface of HLA class I molecule, can be used to detect antibodiesspecific for denatured HLA antigens. In addition, panels of microbeadsthat present denatured class I HLA antigens have been generated and usedto detect antibodies specific for these denatured antigens (See Pei etal. Visuals Clinical Histocompatability Workshop 2000, 9-10).

Currently, those of skill in the art do not consider antibodies specificfor denatured HLA antigens to induce transplant or transfusionrejections. Generally, the presence of denatured HLA antigens isconsidered an artifact of sample preparation and detection of antibodiesspecific for these denatured antigens is not considered a clinicallysignificant step of HLA tissue-typing analysis. One reason for this viewis that cell-based assays are the current standard assay forcross-matching, and there are no current assays available for detectingantibodies specific for denatured antigens alone. As virtualcross-matching and solid-phase assays become standard practices intissue-typing laboratories, there is a need to develop assays thatscreen for antibodies specific for HLA antigens while determiningwhether the antibodies are specific for native or denatured antigens. Inaddition, there is a need to decrease the number of false positive andfalse negative results of both solid-phase and cell-based tissue typingassays.

SUMMARY OF INVENTION

The invention provides for methods of screening for HLA antibodies whichcomprise preparing a panel of solid-phase substrates, wherein eachsubstrate presents at least one selected native HLA antigen or at leastone selected denatured HLA antigen. Even though the art provides formethods of detecting antibodies specific for denatured HLA antigens andit is likely that panels of HLA antigens currently used in the artpresent some denatured antigens mixed with native antigens, the need todistinguish between antibodies specific for denatured HLA antigens andnative HLA antigens was not recognized. The present invention is thefirst to provide methods of screening for antibodies specific for HLAantigens using solid-phase substrates that present a selected native HLAantigen or a selected denatured HLA antigen, and is the first methodwherein the denatured HLA antigens are separated from native HLAantigens and concentrated on a substrate. The clinical significance ofantibodies specific for denatured HLA antigens was unknown and thereforethe available screening methods did not focus on selectively detectingthese antibodies.

It is important to screen for antibodies that specifically bind todenatured antigens, because there is a chance that a transplantrecipient may have a high reactivity to a denatured HLA antigen and maynot have the same reactivity for the antigen in its native conformation.Cell-based screening assays are unlikely to detect the presence ofantibodies that specifically bind to denatured HLA antigens becausethere is minimal presentation of denatured antigens on the cell surface.Failure to detect a high reactivity to a denatured HLA antigen increasesthe risk for rejection of the transplanted organ. Therefore, screeningfor denatured antigens is an important step in fully analyzing atransplant recipient for HLA antigen reactivity prior to transplant.

In addition, many laboratories are carrying out virtual cross-matchingassays and solid-phase assays in conjunction with standard cell-basedassays. For virtual cross-matching to be effective, the HLA antibody andantigen profiles of the transplant or transfusion recipients and donorsshould include information regarding denatured HLA antigens andantibodies thereto. Antibodies to denatured HLA antigens have beendetected in untransfused transplant recipients. These findings suggestthat the cell-based assays may be missing clinically significantantibodies to denatured HLA antigens and these antibodies may play arole in previously unexplained transplant rejections. On the other hand,detection of serum reactivity due to antibodies that specifically binddenatured HLA antigens rather than native antigens in a virtualcross-matching assay may be a false positive result that would not bedetected in a cell-based assay. These false positive results maynegatively affect transplant organ allocation by labeling a transplantpatient ineligible due to an inaccurate HLA antibody profile.

The methods of the invention may be carried out with any type of HLAantigen including class I and class II HLA antigens. The term “nativeHLA antigen” refers to a HLA antigen that is in a conformation that is astable three-dimensional structure that determines the protein'sbiological function. A native HLA class I molecule consists of a 45 kDaglycoprotein which is non-covalently associated with β2-microglobulin(β2m). A native HLA class II molecule consists of the antigen in aheterodimeric conformation. The term “denatured HLA antigen” refers to aHLA molecule that is not in its native conformation. A denatured HLAclass I antigen is disassociated from β2m and a denatured HLA class IIantigen is in a monomeric conformation.

In one embodiment, the invention provides for methods of screening forantibodies that specifically bind native HLA antigens or denatured HLAantigens, comprising the steps of: a) preparing a panel of solid-phasesubstrates, wherein the panel comprises substrates that present at leastone selected native HLA antigen and substrates that present at least oneselected denatured HLA antigen, b) obtaining a serum sample from a humansubject, c) contacting each panel with the serum sample and f) detectingbinding of an antibody to the panel, wherein binding of an antibody to asolid-phase substrate presenting a denatured HLA antigen is indicativeof a human subject having antibodies specific for a selected denaturedHLA antigen, and wherein binding of an antibody to a solid-phasesubstrate presenting a selected native HLA antigen is indicative of ahuman subject having antibodies specific for a selected HLA antigen.

The term “selected HLA antigen” refers to intentionally presenting aparticular HLA antigen on the solid-phase substrate. Unlike currentmethods in the art, the invention provides for intentionally presentingparticular denatured HLA antigens or particular native HLA antigens onsolid-phase substrates in order to distinguish whether a donor hasantibodies that specifically bind to denatured HLA antigens, native HLAantigens or both.

The term “selected denatured HLA antigens” refers to intentionallypresenting a particular HLA antigen on a solid-phase substrate andintending to denature the antigen either while associated with thesolid-phase substrate or prior to being associated to the solid-phasesubstrate. In preferred embodiments, the treatment to denature the HLAantigen will cause all or predominantly all of the HLA antigenspresented on the solid-phase substrate to be in a denaturedconformation. Therefore, binding of an antibody to the solid-phasesubstrate presenting a selected denatured antigen is indicative of ahuman subject having antibodies specific for a selected denatured HLAantigen.

The term “selected native HLA antigens” refers to intentionallypresenting a particular native HLA antigen on a solid-phase substrate.It is preferred that the selected native HLA antigens are predominantlyin their native conformation, however, some embodiments may contain amixture of native and denatured antigens. Therefore, an antibody thatbinds to a solid-phase substrate presenting a selected native antigen isindicative of a human subject having antibodies specific for a selectedHLA antigen, which may be in native conformation or in denaturedconformation. Techniques to separate the native and denatured HLAantigens while coated on a solid-phase substrate are one method ofpreparing a solid-phase panel presenting predominantly native HLAantigens.

The invention also provides for methods of screening for antibodies thatspecifically bind HLA antigens in the native conformation, antibodiesthat specifically bind HLA antigens that are denatured and antibodiesthat specifically bind a HLA antigen in both its native and denaturedconformation. These methods involve contacting a serum sample with asolid-phase substrate or cell that present the HLA antigen to absorb theantibodies that bind the HLA antigen in the opposing conformation priorto screening (contacting and detecting binding) for the HLA specificantibodies of interest. For example, when screening for antibodies thatspecifically bind a HLA antigen in its native conformation, the serawill first be contacted to a cell or solid-phase substrate presentingthe HLA antigen that has been denatured in order to remove antibodiesthat specifically bind the denatured HLA antigen to generate a serumsample essentially free of antibodies that specifically bind thedenatured HLA antigen. Alternatively, when screening for antibodies thatbind a HLA antigen in its denatured conformation, the sera will first becontacted to a cell or solid-phase substrate that presents the HLAantigen in its native conformation in order to remove antibodies thatspecifically bind the native HLA antigen to generate a serum sample thatis essentially free of antibodies that specifically bind to said nativeHLA antigens.

In one embodiment, the invention provides for methods of screening forantibodies that specifically bind a native HLA antigen, comprising thesteps of: a) obtaining a serum sample from a human subject, b) providinga first solid-phase substrate having immobilized thereon said HLAantigen which has been denatured, c) binding antibodies thatspecifically bind said denatured HLA antigen in said serum sample tosaid first solid-phase substrate to form an antibody/antigen complex, d)separating the antibody/antigen complex from the serum sample togenerate a serum sample essentially free of antibodies that specificallybind said denatured HLA antigen, e) contacting the serum sample that isessentially free of antibodies that specifically bind said denatured HLAantigen with a second solid-phase substrate presenting the native HLAantigen, and f) detecting binding of an antibody to the secondsolid-phase substrate, wherein binding of an antibody is indicative of ahuman subject having antibodies that specifically bind the native HLAantigen.

In another embodiment, the invention provides for methods of screeningfor antibodies that specifically bind a native HLA antigen, comprisingthe steps of: a) obtaining a serum sample from a human subject, b)providing a cell that presents said HLA antigen which has beendenatured, c) binding antibodies that specifically bind said denaturedHLA antigen in said serum sample to said cell to form anantibody/antigen complex, d) separating the antibody/antigen complexfrom the serum sample to generate a serum sample essentially free ofantibodies that specifically bind said denatured HLA antigen, e)contacting the serum sample that is essentially free of antibodies thatspecifically bind said denatured HLA antigen with a solid-phasesubstrate presenting the native HLA antigen, and f) detecting binding ofan antibody to the solid-phase substrate presenting the native HLAantigen, wherein binding of an antibody is indicative of a human subjecthaving antibodies that specifically bind the native HLA antigen.

The invention also provides for methods of screening for antibodies thatspecifically bind a HLA antigen that has been denatured, comprising thesteps of: a) obtaining a serum sample from a human subject, b) providinga first solid-phase substrate having immobilized thereon said HLAantigen in its native conformation, c) binding antibodies thatspecifically bind said native HLA antigen in said serum sample to saidfirst solid-phase substrate to form an antibody/antigen complex, d)separating the antibody/antigen complex from the serum sample togenerate a serum sample essentially free of antibodies that specificallybind said native HLA antigen, e) contacting the serum sample that isessentially free of antibodies that specifically bind said native HLAantigen with a second solid-phase substrate presenting the HLA antigenthat has been denatured, and f) detecting binding of an antibody to thesecond solid-phase substrate, wherein binding of an antibody isindicative of a human subject having antibodies that specifically bindthe denatured HLA antigen.

In another embodiment, the invention provides for methods of screeningfor antibodies that specifically bind a HLA antigen that has beendenatured, comprising the steps of: a) obtaining a serum sample from ahuman subject, b) providing a cell that presents said HLA antigen in itsnative conformation, c) binding antibodies that specifically bind saidnative HLA antigen in said serum sample to said cell to form anantibody/antigen complex, d) separating the antibody/antigen complexfrom the serum sample to generate a serum sample essentially free ofantibodies that specifically bind said native HLA antigen, e) contactingthe serum sample that is essentially free of antibodies thatspecifically bind said native HLA antigen with a solid-phase substratepresenting the HLA antigen that has been denatured, and f) detectingbinding of an antibody to the solid-phase substrate, wherein binding ofan antibody is indicative of a human subject having antibodies thatspecifically bind the denatured HLA antigen.

The invention further provides for methods of screening for antibodiesthat specifically bind HLA antigens that comprising various permutationsand combination of any of the preceding methods. One of skill in the artwill recognize that any number or combination of the steps can be usedto characterize a serum sample in regards to anti-HLA antibody profiles.For example, it may be of interest to screen a serum sample forantibodies that bind to a HLA antigen in its native conformation, andthen absorb the antibodies that specifically bind to said HLA antigenthat is denatured and then detect if the resulting serum (that isessentially free of antibodies that specifically bind the denatured HLAantigen) continues to bind to a solid-phase panel that present the HLAantigen in its native conformation.

Further, serum samples may comprise antibodies that specifically bind aHLA antigen in its native and denatured conformation. In this example, askilled artisan will understand that the absorption step may strip theserum of transplant relevant antibodies, thus carrying out a combinationof the steps of screening methods is necessary to ensure the propercontrols are run to ensure the results are true positives or truenegatives.

A serum sample may be polyclonal for a particular HLA antigen andtherefore comprise one or more of the following: antibodies that bind aHLA antigen in its native conformation, antibodies that bind said HLAantigen in its denatured conformation and antibodies that bind said HLAantigen in its native and denatured conformation. One of skill in theart will appreciate that the steps of the preceding screening methodscan be combined in different manners to distinguish between sera thatcomprises antibodies that bind a HLA antigen in its native conformation,an antibody that binds a HLA antigen in its denatured conformation or anantibody that binds a HLA antigen in both its native and denaturedconformation.

The absorption step of any of the preceding methods of screening may becarried out with cells that express the HLA antigen in a native ordenatured conformation. The cells may endogenously express the HLAantigen such as platelets or leukocytes. In addition, cells may beengineered to recombinantly express the HLA antigens (in either thenative or denatured conformation) on the cell surface. These cells maynot endogenously express the HLA antigen or express the HLA antigen atlow level. Furthermore, cells that endogenously express the HLA antigenin a native or denatured conformation may be engineered to recombinantlyexpress the HLA antigen at levels greater than endogenous expression.

A solid-phase substrate that presents a HLA antigen in its nativeconformation may also present a HLA antigen in its denaturedconformation as an artifact of sample preparation. However, the numberof denatured antigens on the solid-phase substrate in this situationshould be much lower than the number of native HLA antigens. Thus, whencarrying out the absorption step of any of the preceding screeningmethods, one of skill in the art will appreciate that any effect ofunderlying denatured HLA antigens may be effectively removed bytitrating the volume of test sera and the immunosorbents.

The methods of screening for antibodies that specifically bind nativeHLA antigens and antibodies that specifically bind denatured HLAantigens are useful for characterizing the HLA antibody profiledetermined by solid-phase assays or virtual cross-matching assays. Thesemethods assist in determining whether a result is a true positive, truenegative, false positive or false negative. As more laboratories areusing solid-phase assays and virtual cross-matching, these methods willbe a valuable tool for analyzing the results.

In another embodiment, the invention provides for methods of predictingwhether a transplant recipient has an increased risk for rejection ofthe transplanted organ comprising the steps of: a) determining HLAantibody profile of the recipient using a solid-phase assay comprising apanel of solid-phase substrates wherein the panel comprises substratesthat present at least one selected native HLA antigen and substratesthat present at least one selected denatured HLA antigen, b) determiningHLA antigen profile of the donor of the transplanted organ, c) comparingthe recipient HLA antibody profile and the donor HLA antigen profile,wherein a positive result is when the HLA antigen profile indicates therecipient sera contains an antibody specific for a HLA antigen withinthe donor HLA antigen profile and a negative result is when the HLAantibody profile indicates the recipient sera does not contain anantibody specific for a HLA antigen within the donor HLA antigenprofile, d) cross-matching the recipient sera and the donor tissue,wherein a positive result is when the recipient sera contains antibodiesspecific for a HLA antigen on the donor tissue and a negative result iswhen the recipient sera does not contain antibodies specific for a HLAantigen on the donor tissue, and e) predicting whether the recipient hasan increased risk for rejection of the transplanted organ, wherein anincreased risk for rejection of the transplanted organ is predicted whenthe HLA antibody profile determined by the solid-phase assay is positiveand the cross-match is negative. The invention include methods ofpredicting long term chronic rejection.

The method of predicting whether a transplant recipient has an increasedrisk for rejection may further comprise a step of selecting a donor bycomparing the recipient antibody profile with the donor HLA antigenprofile, wherein the donor antigen profile is determined using asolid-phase assay that comprises a panel of solid-substrates presentingantibodies specific for HLA antigens.

In another embodiment, the invention provides for methods of predictingwhether a transplant recipient has an increased risk for developinggraft vs. host disease comprising the steps of: a) determining HLAantibody profile of the donor of the transplanted organ using asolid-phase assay comprising a panel of solid-phase substrates whereinthe panel comprises substrates that present at least one selected HLAantigen and substrates that present at least one selected denaturedantigen, b) determining HLA antigen profile of the recipient of thetransplanted organ, c) comparing the donor HLA antibody profile and therecipient HLA antigen profile, wherein a positive result is when the HLAantibody profile indicates the donor sera contains an antibody specificfor a HLA antigen within the recipient HLA antigen profile and anegative result is when the HLA antibody profile indicates the donorsera does not contain an antibody specific for a HLA antigen within therecipient HLA antigen profile, d) cross-matching the donor sera and therecipient tissue, wherein a positive result is when the donor seracontains antibodies specific for a HLA antigen on the recipient tissueand a negative result is when the recipient sera does not containantibodies specific for a HLA antigen on the donor tissue, and e)predicting whether the recipient has an increased risk for developinggraft vs. host disease, wherein an increased risk for graft vs. hostdisease is predicted when the HLA antibody profile determined by thesolid-phase assay is positive and the cross-match is negative. Thismethod is preferably carried out wherein the recipient is in need of abone marrow transplant, liver transplant or transplant of any organ thatcontains progenitor stem cells or lymphocytes.

The method of predicting whether a transplant recipient has an increasedrisk for developing graft vs. host disease may further comprise a stepof selecting a recipient by comparing the recipient antigen profile withthe donor HLA antibody profile, wherein the recipient antigen profile isdetermined using a solid-phase assay that comprises a panel ofsolid-substrates presenting antibodies specific for HLA antigens.

The cross-match assay may be carried out using any cell-based antibodydetection assay in known in the art. Exemplary cross-match assays arethe complement-dependent cytotoxicity test (CDC), CDC with antiglobulinaugmentation (AHG) and flow Cytometry Cross-match (FC) (See Noreen, TheAmerican Society for Histocompatibility and Immunogenetics LaboratoryManual, 3rd Ed, I.C.1.1-I.C.I.13).

A “positive” result in a cell-based cross-match assay is when anantibody in the recipient sera binds to a donor cell. In cytotoxiccell-based cros s-match assays, a “positive” result is when therecipient sera is cytotoxic to the donor cells, which indicates the seracontained antibodies specific for the HLA antigens on the donor cells.The FC cross-match assay uses donor cells and recipient sera, however a“positive” result is detected by a fluorescently labeled secondaryantibody that binds to the complex of the recipient antibody binding tothe donor cells. The binding of the secondary antibody causes a channelshift detected by a flow cytometer.

A “negative” result in a cell-based cross-match assay is when therecipient sera does not contain antibodies that bind to the donor cells,which suggest that the recipient will not reject the donor's tissue upontransplantation. In a cytotoxic cell-based cross-match assay, a negativeresult is when the recipient sera is not toxic to the donor cells. Inthe FC cross-match assay, a negative result is when a donorcell/recipient antibody complex is not generated and therefore thesecondary antibody does not bind the complex and a channel shift doesnot occur.

The solid-phase assay or a virtual cross-match assay allows therecipient sera to be tested for antibodies that will bind to HLAantigens presented on a solid substrate and thereby determine the “HLAantibody profile” for the transplant recipient. The donor tissue istyped using serology methods or DNA-based methods to determine thedonor's “HLA antigen profile.” The serology assays use known antibodiesto detect the HLA antigens within a donor tissue, such as themicrolymphocytotoxicity assay, as described in Terasaki et al. Nature204: 998-1000, 1964, which uses serum having known HLA specificity anddonor lymphocytes. DNA-based assays will use PCR or hybridizationmethods to determine the HLA antigens within a donor tissue. Theseassays are considered “virtual” because the recipient sera and donortissue are not contacted and tested directly. A “positive” result of thevirtual cross-match assay or a solid-phase assay is when the recipientHLA antibody profile indicates the presence of a HLA antibody within thesera that is specific for a HLA antigen within the donor's HLA antigenprofile. A “negative” result of the virtual cross-match assay or asolid-phase assay is when the recipient HLA antibody profile indicatesthat the recipient does not have antibodies specific for the HLAantigens within the donor's HLA antigen profile.

The methods of the invention may be carried out with any solid-phasesubstrate. In preferred embodiments, the methods are carried out withsolid-phase panels comprising a set of microparticles or microbeads,wherein each microparticle presents at least one HLA antigen.

The detection steps of the methods of the invention may be carried outwith any technique that recognizes antibody binding to a solid-phasesubstrate. In preferred embodiments, the detection steps are carried outusing a flow cytometer and/or a secondary antibody. Exemplary secondaryantibodies include those which comprise a label selected from the groupconsisting of a radioactive label, fluorescent label, enzymatic label,avidin label or biotin label.

The methods of the invention are carried out with a serum sampleobtained from a human subject. In preferred embodiments, the serumsample is obtained from a transplant or transfusion recipient or atransplant or transfusion donor.

In a further embodiment, the invention provides for kits to carry outthe methods of the invention. The invention provides for kits used toscreen for antibodies that specifically bind to a denatured HLA antigenand/or a native HLA antigen, comprising a panel of solid-phasesubstrates, wherein the panel comprises substrates that present at leastone selected native HLA antigen and substrates that present at least oneselected denatured HLA antigen. In addition, the invention provides forkits used to predict whether a transplant recipient has an increasedrisk of rejecting the transplanted organ comprising a panel ofsolid-phase substrates, wherein the panel comprises substrates thatpresent at least one selected native HLA antigen and substrates thatpresent at least one selected HLA denatured antigen. Then invention alsoprovides for a kit for predicting whether a transplant recipient has anincreased risk of developing graft vs. host disease, comprising a panelof solid-phase substrates wherein the panel comprises substrates thatpresent at least one selected native HLA antigen and substrates thatpresent at least one selected denatured HLA antigen.

When preparing a panel of HLA antigens for screening biological samples,the techniques used to associate HLA antigens to a solid substrateresults in the association of a mixture of native HLA antigens anddenatured HLA antigens. In the past, the clinical significance of thepresence of denatured HLA antigens and antibodies specific for thesedenatured HLA antigens was not recognized, and therefore, the generationof a mixture of native and denatured HLA antigens was not consideredimportant. The teachings herein demonstrate the need to distinguishbetween native and denatured HLA antigens. Therefore, the inventionprovides for methods of removing denatured HLA antigens or native HLAantigens from a sample.

The method of removing denatured HLA antigens from a sample comprise thesteps of: contacting a sample with an antibody specific for denaturedHLA antigens to form a complex comprising the denatured HLA antigen andthe antibody, and separating the complex from the sample, wherein theseparation removes the denatured HLA antigens from the sample. Thismethod may further comprise the step of isolating the native HLA antigenfrom the sample and optionally presenting the native HLA antigen on asolid-phase substrate. The method of removing native HLA antigens from asample, comprises the steps of: contacting the sample with an antibodyspecific for native HLA antigens to form a complex comprising the nativeHLA antigen and the antibody, and separating the complex from thesample, wherein the separation removes the native HLA antigens from thesample.

This method of removing denatured HLA antigens or native HLA antigensmay further comprise the step of isolating from the sample the denaturedor native HLA antigens that remain in the sample after the separation,and optionally presenting these isolated denatured or native HLAantigens on a solid-phase substrate. In one embodiment, the contactingstep of these methods is carried out with an antibody that is bound toan affinity column.

In another embodiment, the invention provides for methods of removingdenatured antigens from a sample comprising contacting the sample with ablocking agent, such as a blocking antibody, wherein binding of thedenatured antigen to the blocking agent removes the antigen from thesample. The blocking effectively results in an antigen that cannot bindto antibodies specific for the antigen within a biological sample, andtherefore only native HLA antigens are available for detection in thescreening assay.

The denatured HLA antigens or native HLA antigens may be removed fromany sample that comprises HLA antigens derived from such as a cellsample including mammalian, insect or bacteria cells expressingrecombinant HLA antigens, tissue sample, blood sample or serum sample.Exemplary samples that contain HLA antigens include cell lysates, tissuelysates, tissue homogenates and cell culture media into whichrecombinant HLA antigen are expressed. The samples of the inventioninclude purified HLA antigens from various sources such as recombinantantigens expressed by bacteria, insect cells or mammalian cells, forexample transformed bacteria may express HLA antigen within inclusionbodies, and the cell lysates or cell culture media containing theseinclusion bodies may be used as a sample for these methods.

The antigens in the samples may be denatured due to sample preparationor may be intentionally denatured prior to the contacting step of themethods. The antigens may be denatured using an acidic denaturing buffersuch as a buffer containing 0.3M Glycine-HCl pH 2.7 with 1% BSA.

The invention also provides for methods of preparing panels ofsolid-phase substrates that present native HLA antigens that are freefrom denatured antigens or panels of solid-phase substrates that presentdenatured HLA antigens that are free from native HLA antigens. Themethods of preparing a panel of solid-phase substrates presenting nativeHLA antigens free from denatured HLA antigens, comprise the steps of:contacting a sample comprising HLA antigens with an antibody that isspecific for denatured HLA antigens to form a complex comprising thedenatured HLA antigen and the antibody, separating the complex from thesample, and isolating the native HLA antigens from the sample andpresenting the native HLA antigens on a solid phase substrate. The term“free from denatured HLA antigens” refers to a solid-phase substratethat predominantly presents native HLA antigens and presents anegligible amount if any denatured HLA antigens.

The methods of preparing a panel of solid-phase substrates presentingdenatured HLA antigens free from native HLA antigens, comprise the stepsof: contacting a sample comprising HLA antigens with an antibody that isspecific for native HLA antigens to form a complex comprising the nativeHLA antigens and the antibody, separating the complex from the sample,and isolating the denatured HLA antigens from the sample and presentingthe denatured HLA antigens on a solid-phase substrate. The term “freefrom native HLA antigens” refers to a solid-phase substrate thatpredominantly presents denatured HLA antigens and presents a negligibleamount if any native HLA antigens.

In one embodiment, the contacting step of these methods is carried outwith an antibody that is bound to an affinity column. The denatured HLAantigens or native HLA antigens may be removed from any sample thatcomprises HLA antigens derived from such as a cell sample includingmammalian, insect or bacteria cells.

BRIEF DESCRIPTION OF DRAWING

FIG. 1A-1B depict a comparison of positive control (PC) serum reactionpattern to control antigen beads (FIG. 1A) vs. denatured antigen beads(FIG. 1B). The Y-axis lists fluorescent intensities of each antigenbeads that indicate the reactivity level. The bead ID and specificity ofeach antigen bead is listed along the X-axis. The reactions are sortedby the reactivity level from strong (left) to weak (right) along theX-axis. PC serum reacted strongly to B27, B7, B49, B67, B81, B42, B56,B57, B58, B53, 63, B8201, B73, and B50 on the control antigen beads;however, it only reacted relative strongly to Cw7 and Cw17 on thedenatured antigen beads.

FIGS. 2A-2B depict a comparison of serum #1 reaction pattern to native(control) HLA antigen beads (FIG. 2A) vs. denatured HLA antigen beads(FIG. 2B). The Y-axis lists fluorescent intensities of each antigen beadthat indicates the reactivity level of the serum to each antigen. Thebeads ID and specificity of each antigen beads is listed along theX-axis. Serum #1 reacted strongly to A30 on both control and denaturedantigen beads.

FIG. 3A-3B depicts a comparison of serum #2 reaction pattern to native(control) HLA antigen beads (FIG. 3A) vs. denatured HLA antigen beads(FIG. 3B). The Y-axis lists fluorescent intensities of each antigen beadthat indicate the reactivity level. The beads ID and specificity of eachantigen beads are listed along the X-axis. Serum #2 reacted to A2, A11,B57 and B58 on the control antigen beads; and it reacted to A2 and B75on the denatured antigen beads.

FIG. 4 depicts that contacting a serum sample with a bead presentingrecombinant denatured HLA A2 antigen resulted in absorption ofantibodies that specifically bind to denatured HLA A2 antigen.

FIG. 5 depicts that contacting a serum sample with a bead presentingrecombinant denatured HLA A1,36 antigen resulted in absorption ofantibodies that specifically bind to denatured HLA A1 antigen.

FIG. 6 depicts that absorption of antibodies that specifically bind todenatured HLA antigens did not affect serum reactivity to native HLAantigens.

DETAILED DESCRIPTION

The experiments described herein suggest that antibodies to denaturedHLA antigens are clinically significant especially for developingcomplete HLA antibody and HLA antigen profiles for virtualcross-matching blood and tissue donors and recipient pairs. Antibodiesto denatured HLA antigens have been detected in untransfused malepatients and in untransfused females who were not known to have beenpregnant. These findings suggest that the cell-based assays may bemissing clinically significant antibodies to denatured HLA antigens.Denatured antigens are not present on cells, so solid-phase assays areimportant for developing assays for denatured HLA antigens.

The experiments described in Example 2 demonstrate that denatured HLAantigens may be separated from native HLA antigens. A panel of β2m-freeHLA heavy chain coated microparticles (denatured antigen beads) can begenerated by treatment of the antigen presenting microparticles with lowpH (as described in Example 1 and taught in Pei et al. (Visuals ClinicalHistocompatability Workshop 2000, 9-10)). Using a denatured antigenpresenting microparticle panel, it was determined for the first timethat multiple polymorphic antibodies against denatured antigens existedin the sera, and the sera also contained antibodies againstβ2m-associated antigens. This multiplex assay allows one to distinguishantibodies against individual specificity of native vs. denaturedantigens. With this tool, the antibodies to the denature antigens can beidentified and their functions can be further studied.

In addition, the experiments described in Example 3 demonstrate thatserum from some patients have a high reactivity to denatured HLAantigens, while these same patients do not have a high reactivity to thecorresponding native HLA antigens. The experiments provide the HLAantigen profile for a positive control serum and two actual patients.The positive control serum has a low reactivity to the denatured HLAantigens (see FIG. 1). In patient sample #1, the serum was highlyreactive to one HLA antigen (A30), the control beads comprise bothdenatured and native antigens so it is unknown whether this sample hasreactivity to both denatured and native antigens (FIG. 2). Therefore,screening for reactivity to denatured antigens in this patient may notbe imperative. However, patient sample #2 was highly reactive to 4native HLA antigens and was highly reactive to 2 different denatured HLAantigens (FIG. 3). The B75 antigen was only be detected in the denaturedantigen panel. Therefore, if patient #2 is not screened for reactivityto denatured antigens, it is likely that this patient will reject thetransplanted organ. Therefore, this study suggests that it is imperativeto screen for both native and denatured HLA antigens.

These experiments demonstrate that not all transplant recipients requirescreening for antibodies to denatured antigens, but some recipients inthe general population, i.e. patient #2 described herein, are at ahigher risk for rejection of a transplanted organ due to theirreactivity to different native and denatured HLA antigens. Thus, thereis a clinical significance to screening for antibodies that are highlyreactive to native and denatured antigens.

Even though most reports relating to anti-HLA denatured antigens focuson the presence of reactivity to denatured class I antigens, theexperiments presented in Example 3 demonstrate that the use ofrecombinantly expressed and purified individual α and β chain HLA classII antigens may be used to detect the presence of antibodies thatspecifically bind denatured HLA class II antigens. This assay allows forthe detection of antibodies specific for denatured HLA class II antigensdespite the lack of well-characterized monoclonal antibodies that allowfor the verification of class II antigen denaturation.

The presence of antibodies that specifically bind to denatured HLAantigens may reduce the sensitivity of solid-phase antibody detectionassays because a serum may have reactivity to denatured HLA antigens ina solid-phase assay but would not react to native HLA antigens in acell-based assay. The reactivity to a denatured HLA antigen would beconsidered a false positive since the transplant recipient is unlikelyto present denatured HLA antigens in vivo. The false positive resultsmay negatively impact transplant tissue allocation because a prospectiverecipient may be turned away due to a false positive result. Theexperiments in Example 4 demonstrate that serum reactivity to denaturedHLA antigens may be reduced or eliminated by absorption of theantibodies that specifically bind denatured HLA antigens usingrecombinantly expressed and purified single chain HLA antigens. Removalof serum reactivity to denatured HLA antigens allows for confirmationand enhanced detection of specific (transplant relevant) antibodies thatbind to exposed epitopes on native HLA antigens (properly foldeddimers). This assay should improve the concordance between solid-phaseantibody tests using purified antigens and cell-based cross-match assaysthereby reducing the number of false positive results. The invention isdescribed in terms relating to detecting HLA specific antibodies andantigens using microbeads. However, the invention can be carried outusing any type of solid-phase substrate known in the art and examples ofsuch solid-phase substrates and assays are described herein.

HLA Antigens

The HLA locus is highly polymorphic in nature. As disclosed in theNomenclature for Factors of the HLA System 2000 (Hum. Immunol.;62(4):419-68, 2001) there are 124 HLA-A alleles, 258 HLA-B alleles, 74HLA-C alleles, 221 HLA-DRB1 alleles, 19 DRB3 alleles, 89 DRB4 alleles,14 DRB5 alleles, 19 DQA1 alleles and 39 DQB1 alleles, with new allelesbeing discovered continuously. As testament to this rapid progress, aApril 2007 update by the WHO nomenclature Committee for Factors of theHLA System (www.anthonynolan.com/HIG/) showed there are 545 HLA-Aalleles, 895 HLA-B alleles, 307 HLA-C alleles, 8 HLA-E alleles, 12 HLA-Halleles, 9 HLA-J alleles, 6 HLA-K alleles, 4 HLA-L alleles, 4 HLA-Palleles, 3 HLA-V alleles, 3 DRA alleles, 494 DRB1 alleles, 1 DRB2alleles, 44 DRB3 alleles, 13 DRB4 alleles, 18 DRB5 alleles, 3 DRB6alleles, 2 DRB7 alleles, 10 DRB8 alleles, 1 DRB9 alleles, 34 DQA1alleles, 83 DQB1 alleles, 23 DPA1, 126 DPB1 alleles, 4 DMA alleles, 7DMB alleles, 12 DOA alleles and 9 DOB alleles.

Methods of Detecting HLA Specific Antibodies

The invention provides for methods of detecting antibodies specific fordenatured HLA antigen, antibodies specific for native HLA antigen andantibodies specific for HLA antigens in both the native and denaturedconformation in a biological sample. The methods are carried out withsolid-phase panels wherein the panel comprises substrates that present(or have immobilized) at least one or more selected native HLA antigensand substrates that present at least one or more selected denatured HLAantigens. Preferably, the HLA antigens are presented on microbeads andare denatured while attached to the microbead. For example, class I HLAantigens can be dissociated from βm2 while on the microbeads using lowpH treatment such as the method provided in Pei et al. (Visuals ClinicalHistocompatability Workshop 2000, 9-10). Class II HLA antigens coated onmicrobeads may be denatured by subjecting the microbeads to low PHconditions such as described in Tampé et al. (Proc Natl Acad Sci USA.,88(23): 10667-10670, 1991). Binding of antibodies to the microbeads maybe detected using methods such as FlowPRA (One Lambda) or Labscreenusing Labscan flow cytometry.

The HLA antigens may be denatured while coated on a solid substrateusing any method known to denatured proteins which will not damage thesolid substrate. Exemplary treatments that will denature proteinsinclude low or high pH, chaotropic agents, such as urea and guanidinehydrochloride at high concentrations such as 4 M to 8 M, detergents suchas SDS and heat. For example, the antigen many be denatured in a buffercontaining 0.3M Glycine-HCl pH2.7 with 1% BSA by mixing a 1:10 ratio ofbead to denaturing buffer.

The invention also may be carried out with liquid-phase assays such asassays using column chromatography, affinity chromatography, thin layerchromatography, liquid-phase immunodiagnostic (LIPA) assays,liquid-phase chemiluminescent ELISA and liquid-phase immunoradiometric(IRMA) to name a few. The HLA antigens may be denatured while in theliquid phase or prior to being inserted into the liquid-phase assay.

Antigens of the invention may be a whole protein, a truncated protein, afragment of a protein or a peptide. Antigens may be naturally occurring,genetically engineered variants of the protein, or may be codonoptimized for expression in a particular mammalian subject or host.Generally, a B-cell epitope will include at least about 5 amino acidsbut can be as small as 3-4 amino acids. The antigens may berecombinantly expressed and purified from cells that either endogenouslyexpress the HLA antigens at a low level or do those that do notendogenously express the HLA antigens. Furthermore, the HLA antigens maybe recombinantly expressed and presented on the cell surface, and thecells would be used in the methods of the invention.

Normally, an epitope will include between about 7 and 15 amino acids,such as, 9, 10, 12 or 15 amino acids. The term “antigen” denotes bothsubunit antigens, (i.e., antigens which are separate and discrete from awhole organism with which the antigen is associated in nature).Antibodies such as anti-idiotype antibodies, or fragments thereof, andsynthetic peptide mimotopes, that is synthetic peptides which can mimican antigen or antigenic determinant, are also captured under thedefinition of antigen as used herein.

Furthermore, for purposes of the present invention, an “antigen” refersto a protein, which includes modifications, such as deletions, additionsand substitutions, generally conservative in nature, to the naturallyoccurring sequence, so long as the protein maintains the ability toelicit an immunological response, as defined herein. These modificationsmay be deliberate, as through site-directed mutagenesis, or may beaccidental, such as through mutations of hosts which produce theantigens. Antigens of the present invention may also be codon optimizedby methods known in the art to improve their expression orimmunogenicity in the host.

Exemplary solid-phase assays such as assays of the invention may usesolid substrates such as microparticles, magnetic particles such asferromagnetic beads and paramagnetic beads, microtiter plates,membranes, filters, glass, metal, metal-alloy, anopol, polymers, nylon,plastic or microarrays such as protein chips. Microarrays may be of anymaterial such as glass or silica. Binding on a microtiter plate may bedetected using ELISA assays, RIA assays or other immunosorbent sandwichassays. Binding on a filter may be detected using immunoblottingtechniques.

The solid-phase assays of the invention may be carried out withmicroparticles, microbeads, magnetic beads, beads or microspheres of anymaterial, e.g. silica, gold, latex, polymers such as polystyrene,polysulfone and polyethyl, or hydrogel. Additional exemplarymicroparticles are encoded with the dyes and the antigens areimmobilized to the encoded microparticles. The microparticles used inthe methods of the invention are commercially available from sourcessuch from Luminex Inc., Invitrogen (Carlsbad, Calif.), Polysciences Inc.(Warrington, Pa.) and Bangs Laboratories (Fishers, Ind.) to name a few.

The microparticles of the invention may comprise a detectable label oranother identifying characteristic. The microparticles may comprise asingle fluorescent dye or multiple fluorescent dyes. In one embodiment,the microparticles are internally labeled with fluorescent dyes andcontain surface carboxyl groups for covalent attachment of biomolecules.In another embodiment, the microparticles are internally labeled withfluorescent dyes and contain a surface layer of Avidin for near covalentbinding of biotin and biotinylated ligands. In another embodiment, themicroparticles may comprise a combination of different dyes, such as afluorescent and a non-fluorescent dye. For example, the microparticlesmay be labeled with E)-5-[2-(methoxycarbonyl)ethenyl]cytidine, which isa nonfluorescent molecule, that when subjected to ultraviolet (UV)irradiation yields a single product,3-13-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine, which displays astrong fluorescence signal. In another embodiment, the microparticlesmay comprise bar codes as an identifiable characteristic as described inU.S. Patent Publication No. US 20070037195.

In another embodiment, the microparticles may be nanocrystals or quantumdots. These nanocrystals are substances that absorb photons of light,then re-emit photons at a different wavelength (fluorophores). Inaddition, additional florescent labels, or secondary antibodies may beconjugated to the nanocrystals. These nanocrystals are commerciallyavailable form sources such as Invitrogen and Evident Technologies(Troy, N.Y.),

The invention can be carried out with any system that detects theidentifiable characteristic or label, such as FLOW cytometry. Detectionof fluorescent labels may also be carried out using a microscope orcamera that will read the image on the microparticles, such as theBioarray BeadChip (Bioarray Solutions, Ltd., Warren, N.J.). The BeadChipformat combines microparticle (“bead”) chemistry with semiconductorwafer processing in which binding to the microparticle is recorded usingan optical microscope and camera.

Biological samples includes whole blood, blood derivatives, red bloodcell concentrates, plasma, serum, fresh frozen plasma, whole bloodderived platelet concentrates, apheresis platelets, pooled platelets,intravenous gamma-globulin, cryoprecipitate, cerebrospinal fluid,tissues and cells such as epithelial cells, such as those collected fromthe buccal cavity, stem cells, leukocytes, neutrophils and granulocytes.The biological samples may be obtained from a human donor of tissue orcells intended for transplantation or a human donor of blood or bloodderivatives intended for transfusion. The biological sample may beobtained from a healthy bone marrow donor or a subject of a paternitytest. The biological sample may also be obtained from a human subjectthat is an intended recipient of a transplant or transfusion, or thehuman subject that is donating the tissue or organ intended fortransplantation or transfusion. Alternatively, the biological sample maybe obtained directly from tissues or cells that are intended fortransplantation in a human recipient. In addition, the biological samplemay be obtained from blood or blood derivatives that are intended fortransfusion in a human recipient.

Methods of Detecting HLA Antigens

The invention provides for methods of detecting denatured HLA antigensin a biological sample. Antibodies specific for denatured HLA antigensare used to detect the presence of HLA antigens in a biological sample.Commercial antibodies that are specific for denatured HLA class Iantigens include HB 10 (ATCC) and HB-296 (ATCC).

The antibodies of the invention may be polyclonal antibodies, monoclonalantibodies, antibody fragments which retain their ability to bind theirunique epitope (e.g., Fv, Fab and F(ab)2 fragments), single chainantibodies and human or humanized antibodies. Antibodies may begenerated by techniques standard in the art using an antigenic HLAepitope. See, e.g. Kohler et al., Nature, 256:495-497 (1975), Brodeur etal., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987). Antibody molecules of thepresent invention include the classes of IgG (as well as subtypes IgG 1,IgG 2a, and IgG2b), IgM, IgA, IgD, and IgE.

The antibodies of the invention may be labeled for detection of bindingwithin the biological sample. The antibodies may comprise a radioactivelabel such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I. In addition, the labels may bea fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, phycoerythrin, rhodamine, or luciferin. The labels maybe enzymes such as alkaline phosphatase, β-galactosidase, biotin andavidin or horseradish peroxidase (Bayer et al., Meth. Enz., 184:138-163(1990)).

The antibodies specific for denatured HLA antigens may be attached tosolid substrates such as membranes, beads, filters, glass, silicon,metal, metal-alloy, anopore, polymeric, nylon or plastic for detectionof HNA-3 in a biological sample.

Specific binding of an antibody to a denatured HLA antigen within abiological sample may be carried out using Western blot analysis withimmunoblotting, immunocytochemistry, immunohistochemistry, dot blotanalysis, flow cytometry, ELISA assays or RIA assays. These techniquesand other approaches are conventional in the art (See Sambrook et al.,Molecular Cloning: A Laboratory Manual, cold Springs Harbor Laboratories(New York, 1989).

Kits

The invention also provides for kits to carryout the methods of theinvention. In particular, the invention provides for kit for conductinga method of screening for antibodies specific for native and denaturedHLA antigens. The antigens provided in the kit may be conjugated tosolid substrates in the kit. Alternatively, the kit comprises solidsubstrates and antigens and the skilled artisan can conjugate theantigens to the solid substrates allowing for optimization of theantigens used in the assay. The kits may also comprise the reagentsnecessary to detect and measure antibodies, such as HLA antibodies foruse as a positive control.

Kits according to the invention also may comprise components fordetecting denatured HLA antigens and native HLA antigens in a biologicalsample. The kit can comprise an antibody that specifically binds todenatured HLA antigens. The antibodies provided in the kit may beconjugated to solid substrates in the kit. Alternatively, the kitcomprises solid substrates and antibodies and the skilled artisan canconjugate the antibodies to the solid substrates allowing foroptimization of the antibodies used in the assay. The kit may alsocomprise antigens to use as a positive control. The kits may alsocomprise the reagents necessary to detect and measure HLA antigens in abiological sample.

Kits useful for detecting denatured HLA antigens or antibodies specificfor denatured HLA antigens may further comprise any components necessaryto carry out the detection assays that are conventional in the art. Forexample, the kits may comprise buffers, loading dyes, gels such aspolyacrylamide gels and molecular weight markers for preparing SDS-PAGEgels to carry out Western blots. The kits may also comprise filters,membranes blocking buffers, control buffers, isotype control antibodies,wash buffers or buffers and reagents for detection to carry outimmunoblotting or dot blotting analysis such as labeled secondaryantibodies. The kit may also comprise fixing reagents, blocking buffers,control buffers, wash buffers, staining dyes and detection reagentsincluding anti-idio specific antibodies. Furthermore, the kits maycomprise the necessary reagents and tools to carryout flow cytometry,ELISA assays, RIA assays or microtoxicity assays.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative example.

EXAMPLES Example 1 Method of Denaturing HLA Class I Antigens on Beads

To investigate the presence of antibodies specific for denatured HLAantigens, beads known to present native HLA antigens were subjected todenaturing conditions. FlowPRA Class I beads (FL1-30, FL1SP, One LambdaInc.), Flow Single antigen beads (FL1HD, One Lambda Inc.), LABCreenclass I PRA (LS1PRA, One Lambda Inc.) or LABCreen Single antigen beads(LS1A01, LS1A02, LS1A03, or LS1A04, One Lambda Inc.) were incubated witha pH 3 buffer containing 0.1 M sodium acetate for 30 minutes at roomtemperature. The beads were subsequently washed twice with PBS andresuspended in PBS.

Monoclonal antibodies BIH I (One Lambda, Inc.) and W6/32 (ATCC) areknown to specifically bind to β2m-associated class I antigens and notdenatured HLA antigens. Monoclonal antibodies HC10 (ATCC) or HB-296(ATCC) are known to specifically bind β2m-free class I antigens(denatured HLA antigens) (Perosa et al., J. Immunol. 171: 1918-1926,2003).

To confirm that low pH treated beads were denatured, a panel of treatedbeads were mixed with a panel of untreated (native) beads. These beadwere incubated with the BIH I antibody or the W6/32 antibody.Biotinylated antibody (2 μg) in of PBS (95 μl) were incubated with theantigen presenting beads (5 μl) with or without acid treatment for 30minutes at 20-25° C. with gentle rotation. The microbeads were washed 3times with 1 ml wash buffer (One Lambda., Inc.) by centrifugation at9000 g for 2 minutes and then incubated with 100 μl FITC orPE-conjugated Streptavidin (Jackson ImmunoResearch). The microbeads werethen washed twice with 1 ml wash buffer, and then 0.5 ml fixing solution(PBS with 0.5% formaldehyde) was added to the beads. The beads were thenanalyzed by a flow cytometer or a Luminex machine.

The fluorescent intensity of the antibody binding to the bead indicatesthe reactivity level of the antibodies to either native or denaturedantigens. This analysis demonstrated that the BIH I and the W6/32antibodies did not significantly bind to the beads presenting thedenatured HLA antigens. This experiment demonstrates that the low pHtreatment denatured the HLA antigens presented on beads that arecurrently used for bead-based HLA screening.

Example 2 Separation of β2m-Free (Denatured) and β2m-Associated (Native)HLA Class I Antigens

It was of interest to develop techniques for separating denatured andnative HLA antigens. A panel of untreated HLA antigen-presenting beadswas incubated with an antibody specific for native HLA antigens (W6/32)and an antibody specific for denatured HLA antigens (HB-296; ATCC). Theantigens on the beads were purified from human cell lines, and the beadscontain both β2m+ and β2m-free antigens because β2m may be disassociatedfrom class I HLA antigens in vitro during antigen purification or othermanipulation.

The denatured HLA antigens of the above-identified panel were separatedfrom the native HLA antigens using HC10 affinity column absorption. HC10is an antibody that is specific for denatured HLA antibodies. Mixture ofnative and denatured HLA class I antigens were diluted to about 10-50μg/ml and run through an HC10 affinity column at a flow rate of 0.5ml/min. The native antigen remained in the flow though and the denaturedantigen bound to the HC10 affinity column. The denatured antigens werethen further eluted from the HC10 affinity column with a high or low pHbuffer (pH 11 or pH 3).

Using these techniques, β2m-associated and β2m-free antigens wereseparated. Separation of the denatured HLA antigens allows for selectivescreening of antibodies specific for denatured antigens. Thesetechniques demonstrated that the mixture of β2m-associated and β2m-freeantigens can be separated, which allows for the native and denaturedantigens to be distinguished. In addition, this separation techniqueallows beads to present either denatured or native antigens.

Example 3 Screening HLA Antibodies with Antigen Beads

Sera samples from patients waiting for kidney transplant were screenedfor the presence of antibodies specific for denatured HLA antigens usingpanels of beads which present both native HLA antigens and denatured HLAantigens. Twenty microliters of serum were incubated with 5 μl of eachof the antigen beads with or without acid treatment (as described inExample 1) for 30 minutes at 20-25° C. with gentle rotation. Themicrobeads were washed 3 times with 1 ml wash buffer (One Lambda., Inc.)by centrifugation at 9000 g for 2 minutes and then incubated with 100 μlFITC or PE-conjugated F(ab)2 fragment of goat anti-human IgG (JacksonImmunoResearch). The microbeads were then washed twice with 1 ml washbuffer, and then 0.5 ml fixing solution (PBS with 0.5% formaldehyde) wasadded to the beads. The beads were then analyzed by a flow cytometer ora Luminex machine.

FIG. 1 depicts a comparison of positive control (PC) serum reactionpattern to control antigen beads (FIG. 1A) vs. denatured antigen beads(FIG. 1B). PC serum reacted strongly to HLA antigens B27, 7, 49, 67, 81,42, 56, 57, 58, 53, 63, 8201, 73, and B50 on the control antigen beads,which were not treated with low pH and therefore present both native anddenatured HLA antigens. However, the PC serum only reacted relativestrongly to antigens Cw7 and Cw17 on the denatured antigen beads, whichwere treated with low pH.

FIG. 2 depicts a comparison of serum #1 reaction pattern to controlantigen beads (FIG. 2A) vs. denatured antigen beads (FIG. 2B) asdescribed above. Serum #1 reacted strongly to HLA antigen A30 on bothcontrol (which contains both native and denatured antigens) anddenatured antigen beads. The sample reacted strongly to the A30 antigenon the control beads that was a the mixture of HLA antigens in nativeand denatured conformation, and it reacted strongly with the beads thatcontained antigens in completely denatured conformation, indicating thispatient contains antibodies to denatured A30 antigen.

FIG. 3 depicts a comparison of serum #2 reaction pattern to controlantigen beads (FIG. 3A) vs. denatured antigen beads (FIG. 3B) asdescribed above. Serum #2 reacted to HLA antigens A2, A11, B57 and B58on the control antigen beads; and it reacted to A2 and B75 on thedenatured antigen beads. This sample has a strong reactivity to bothnative and denatured HLA antigens and these HLA antigens were different.Therefore, this assay suggests that failing to screen for reactivity todenatured antigens in addition to native antigens may result inrejection of the transplanted organ.

In addition, the data presented in FIGS. 2 and 3 demonstrate that thesera from actual patients contained antibodies specific for denaturedantigens. Therefore, in view of current transplant rejection rates andthe presence of antibodies specific for denatured HLA antibodies, it islikely that the denatured HLA antigens are clinically significant.

Example 4 Detection of Antibodies Reacting with denatured Class II HLAAntigens

Little is known about antibodies specific for denatured class IIantigens, and there are no well characterized monoclonal antibodies thatspecifically bind to denatured class II HLA antigens and can verify thecompletion of Class II antigen denaturation. The present experimentdescribes an assay to identify antibodies that specifically bind todenatured class II HLA antigens in patient sera.

Individual α and β chain of HLA-DR class II antigens were expressed andpurified from Escherichia coli. The purified single antigen chains wereconjugated onto Luminex beads. Sera samples from patients were screenedfor the presence of antibodies specific for denatured class II HLAantigens using the LABScreen Single Antigen assay standardmanufacturer's protocol (One Lambda, Inc.) and compared to the specialtybeads and LSA01 Single Antigen beads. Table 1 shows the serum reactivityagainst denatured DRα chain (DRA*0101) and DRβ chain (DRB4*0101). Thepositive control was a known antibody that specifically binds to nativeHLA class II antigens. The negative control was a sample that did notcontain any antibodies. The underlined mean fluorescence values (MFI)indicate positive binding by an antibody in the serum. Out of 8 pan-DRreacting serum, all had reactivity to DR β chain and 3 out of 8 seraalso displayed reactivity to DRα chain.

TABLE 1 Allele DRA*0101 DRB4*0101 Concentration of sera 1 μg 2 μg 1 μg 2μg Positive control 3489 3007  3202  3270 Negative control 1148 1041 2636  2934 Serum 1 9417 9426 14239 14085 Serum 2 20833  19307  2452224078 Serum 3 4681 4921 10282 10050 Serum 4 15335  15038  20891 19527Serum 5 4231 4501  9739  9595 Serum 6 4958 5450 20843 20690 Serum 7 44665118 15633 16844 Serum 8 6063 6242 22469 23439 Serum 9 17169  18207 24182 23894 Serum 10 4220 4057 11174 11308 Serum 11 10090  9897 1966219279Table 2 shows the serum reactivity against denatured HLA-DR α chain(DRA*0101), HLA-DR β chain (DRB4*0101), HLA-DP α chain (DPA1*0103),HLA-DP β chain (DPB1*0101) and HLA-DQ β chain (DQB1*0201). Theunderlined Mean Fluorescent Values (MFI) indicates the positivereactions. The sera set out in Table 2 were known to be reactive to selfalleles, and 2 of the 7 serum samples had reactivity to the HLA-DR βchain, HLA-DQ β chain and HLA-DP β chain.

TABLE 2 Allele DRA*0101 DRB4*0101 DPA1*0103 DPB1*0101 DQB1*0201 Conc. 1μg 2 μg 1 μg 2 μg 1 μg 2 μg 1 μg 2 μg 1 μg 2 μg Negative  526  376  1202 1472  665  701 1272  885 1867 1882 control Positive  3236  2306  4877 5701 1893 2249 3525 2814 3855 4160 control Serum 12  702  629  642  6941534 1896 3107 2804 3385 3651 Serum 13  2547  1981  3045  3516 2138 22573948 3144 5225 5516 Serum 14 15890 11447 16350 16892 7845 7630 10982 7653 13390  13447  Serum 15  3207  3156  3896  4581 4528 5000 6496 56396280 6971 Serum 16 12149  8872 12966 13321 6824 6458 7638 5749 9222 9161Serum 17  6754  4479 10582 11733 5805 6944 9962 9478 11137  12212  Serum18  3397  2578  4301  4638 2564 2767 4722 3720 6731 6671 Anti-HlgG   15  15   15   14  12  14  11  10  10  23

Example 5 Absorption of Antibodies Against Denatured HLA Antigens

HLA Class I heavy chain and Class II α and β chain were expressed andpurified from E. coli. The purified single chain antigens wereconjugated onto paramagnetic particles (Spherotech, CM-025-10). Theantigen coated beads were then incubated with test serum (known to reactto denatured HLA antigens) for 30 minutes at room temperature. The beadparticles were then separated from the serum using a magnetic fieldaccording to the manufacturer's instructions. The reactivity of theserum then was assayed using LABScreen Single Antigen beads (One LambdaInc. LS1A04) using the standard manufacturer's protocol.

Serum sample 38 is known to contain antibodies which specifically bindto denatured HLA-A2 antigen. Serum 38 reactivity was tested for thefollowing HLA Class I antigens for A2 (RA0101, RA 201, RA203, RA0206),A11 (RA 1101, RA1102), A23 (RA2301), A24 (RA2402, RA2403), A25 (RA 2501)A26 (RA2601), A29 (RA2901, RA2902), A30 (RA 3001, RA3002), A31 (RA3101),A32 (A3201), A33 (RA3301, RA3302), A34 (A3401, A3402), A36 (RA3601), A68(RA6802), A80 (RA8001), B13/B4 (RB1302), B62/BW6 (RB1501), B71/B6(RB1510), B27/BW6 (RB2708), B38/BW4 (RB380)1, B61/BW6 (RB4002), B46/BW6(RB4601), B49/BW4 (RB4901), B51/BW4 (RB5102), B59/BW4 (RB5901), B78/B6(RB7801), CW1 (RC0102), CW9 (RC0303), CW5 (RC0501), CW8 (RC0801), CW15(RC1501), CW18 (RC1802), B61/BW6 (RB4006). Serum 38 was contacted withbeads presenting purified HLA Class I heavy chain RA0101(A*0101), RA0201(A*0201), RB0702 (B*0702) and denatured HLA-A2 antigen for absorption ofantibodies specific for denatured HLA antigens. Only contacting withbeads presenting A*0201 removed serum reactivity to Class I antigens:RA0201, RA0203, RA 0206, RA2301, RA2402 and RA3401 as shown in FIG. 4.

Serum sample 37 is known to contain antibodies which specifically bindto denatured HLA-A1,36. Serum 37 reactivity was tested for the HLA ClassI antigens listed above. Serum 37 was contacted with beads presentingpurified HLA Class I heavy chain RA0101(A*0101), RA0201 (A*0201), RB0702(B*0702) and denatured HLA-A1,36 for absorption of antibodies specificfor denatured HLA antigens. Only contacting with beads presenting A*0101removed serum reactivity to Class I antigens: RA0101 and RA-3601 asshown in FIG. 5.

FIGS. 4 and 5 are examples demonstrating that denatured HLA antigencoated magnetic beads remove all the antibody reactivity to the HLAantigens that share epitopes with the denatured HLA antigens used forabsorption. This absorption process is antigen specific and did notremove antibody reactivity to native HLA antigens as shown in FIG. 6.specifically, serum 39 does not contain antibodies that specificallybind to denatured HLA antigens. Serum 39 reactivity was tested for theHLA Class I listed antigens listed above. Serum 39 was contacted withbeads presenting purified HLA Class I heavy chain RA0101(A*0101), RA0201(A*0201), RB0702 (B*0702) and denatured antigens for absorption ofantibodies specific for denatured HLA antigens. The serum reactivity tonative antigens was not affected when the serum was absorbed withdenatured A1, A2 or B7 coated beads.

This analysis demonstrates that serum reactivity to denatured HLAantigens can be significantly reduced, if not eliminated, by absorptionwith recombinant single chain HLA antigens, which permits confirmationand enhanced detection of specific (transplant relevant) antibodies thattarget exposed epitopes on native HLA antigens (properly foldeddimmers). This approach should improve the concordance betweensolid-phase antibody detection assays using purified HLA antigens andcell-based cross-matched assays, and thereby reduce false positiveresults determined by the solid-phase assays.

Numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the art upon consideration ofthe presently preferred embodiments thereof. Consequently, the onlylimitations which should be placed upon the scope of the invention arethose which appear in the appended claims.

What is claimed:
 1. A method of screening for antibodies that specifically bind a native HLA antigen, comprising the steps of: obtaining a serum sample from a human subject, providing a first solid phase substrate having immobilized thereon said HLA antigen which has been denatured, binding antibodies that specifically bind said denatured HLA antigen in said serum sample to said first solid-phase substrate to form an antibody/antigen complex, separating the antibody/antigen complex from the serum sample to generate a serum sample essentially free of antibodies that specifically bind said denatured HLA antigen, contacting the serum sample that is essentially free of antibodies that specifically bind said denatured HLA antigen with a second solid-phase substrate presenting the native HLA antigen, and detecting binding of an antibody to the second solid-phase substrate, wherein binding of an antibody is indicative of a human subject having antibodies that specifically bind the native HLA antigen.
 2. A method of screening for antibodies that specifically bind a native HLA antigen, comprising the steps of: obtaining a serum sample from a human subject, providing a cell that presents said HLA antigen which has been denatured, binding antibodies that specifically bind said denatured HLA antigen in said serum sample to said cell to form an antibody/antigen complex, separating the antibody/antigen complex from the serum sample to generate a serum sample essentially free of antibodies that specifically bind said denatured HLA antigen, contacting the serum sample that is essentially free of antibodies that specifically bind said denatured HLA antigen with a solid-phase substrate presenting the native HLA antigen, and detecting binding of an antibody to the solid-phase substrate presenting the native HLA antigen, wherein binding of an antibody is indicative of a human subject having antibodies that specifically bind the native HLA antigen.
 3. The method of claim 1 or 2, wherein the HLA antigen is a HLA class I antigen.
 4. The method claim 1 or 2, wherein the HLA antigen is a HLA class II antigen.
 5. The method of claim 1 or 2, wherein a solid-phase substrate is selected from microparticles, microbeads, magnetic beads and an affinity purification column.
 6. The method of claim 1 or 2, wherein the serum sample is obtained from a human subject that is a transplant or transfusion recipient.
 7. The method of claim 1 or 2, wherein the serum sample is obtained from a human subject that is a transplant or transfusion donor.
 8. A method of screening for antibodies that specifically bind a HLA antigen that has been denatured, comprising the steps of: obtaining a serum sample from a human subject, providing a first solid-phase substrate having immobilized thereon said HLA antigen in its native conformation, binding antibodies that specifically bind said native HLA antigen in said serum sample to said first solid-phase substrate to form an antibody/antigen complex, separating the antibody/antigen complex from the serum sample to generate a serum sample essentially free of antibodies that specifically bind said native HLA antigen, contacting the serum sample that is essentially free of antibodies that specifically bind said native HLA antigen with a second solid-phase substrate presenting the HLA antigen that has been denatured, and detecting binding of an antibody to the second solid-phase substrate, wherein binding of an antibody is indicative of a human subject having antibodies that specifically bind the denatured HLA antigen.
 9. A method of screening for antibodies that specifically bind a HLA antigen that has been denatured, comprising the steps of: obtaining a serum sample from a human subject, providing a cell that presents said HLA antigen in its native conformation, binding antibodies that specifically bind said native HLA antigen in said serum sample to said cell to form an antibody/antigen complex, separating the antibody/antigen complex from the serum sample to generate a serum sample essentially free of antibodies that specifically bind said native HLA antigen, contacting the serum sample that is essentially free of antibodies that specifically bind said native HLA antigen with a solid-phase substrate presenting the HLA antigen that has been denatured, and detecting binding of an antibody to the solid-phase substrate, wherein binding of an antibody is indicative of a human subject having antibodies that specifically bind the denatured HLA antigen.
 10. The method of claim 8 or 9, wherein the HLA antigen is a HLA class I antigen.
 11. The method claim 8 or 9, wherein the HLA antigen is a HLA class II antigen.
 12. The method of claim 8 or 9, wherein a solid-phase substrate is selected from microparticles, microbeads, magnetic beads and an affinity purification column.
 13. The method of claim 8 or 9, wherein the serum sample is obtained from a human subject that is a transplant or transfusion recipient.
 14. The method of claim 8 or 9, wherein the serum sample is obtained from a human subject that is a transplant or transfusion donor. 